- 學位論文
以電子自旋共振光譜儀探討膽固醇對類澱粉前驅蛋白穿膜區域結構之影響
Studying the Effect of Cholesterol on the Structure of Membrane-spanning Region of Amyloid Precursor Protein by Electron Spin Resonance Spectroscopy
摘要
百分之九十以上的阿茲海默症 (Alzheimer’s disease, AD) 屬於偶發性的案例，缺乏顯著的基因遺傳，老化、高血壓、高膽固醇與糖尿病等是目前已知的非基因危險因子。目前的研究發現，患者腦中最初的病理特徵來自乙型類澱粉胜肽 (Amyloid-β, Aβ) 聚集，此胜肽由稱為類澱粉前驅蛋白 (amyloid precursor protein, APP) 的第一型膜蛋白經β 分泌酶 (β-secretase) 與 γ 分泌酶 (γ-secretase) 作用產生。然而，γ 分泌酶缺乏專一的剪切區域，因此產生 C 端序列不同的乙型類澱粉胜肽。這些長度不一的胜肽具有不同聚集能力與細胞毒性，其中 42 個胺基酸組成的乙型類澱粉胜肽有較強的聚集能力，容易聚集在患者的腦中，造成毒性。腦中膽固醇含量是影響非遺傳性阿茲海默症的危險因子之一，我們認為膽固醇的存在可能影響類澱粉前驅蛋白穿膜區域的結構，進而改變 γ 分泌酶的切位。因此以固相合成法合成包含類澱粉前驅蛋白穿膜區域序列的胜肽 (KKWK-Aβ22-55)，並在特定點位置將殘基取代為半胱氨酸，以利於含有自由基的化合物 MTSSL 標記，最後將這些胜肽分別鑲嵌在微脂體中，透過包含 double electron-electron resonance (DEER)、electron spin echo envelope modulation (ESEEM) 及 continuous-wave electron spin resonance (CW-ESR) 在內的電子自旋共振光譜技術分析這些自旋標記間的距離變化。本研究結果顯示膽固醇的加入會降低類澱粉前驅蛋白穿膜區域的結構彈性，同時增加穿膜區域結構長度；另外 36-40 片段由胺基酸序列 VGGVV 組成，包含兩個相連的甘胺酸卻依然維持 α-螺旋結構，並未產生甘胺酸扭結 (GG kink) 而形成 310 螺旋，此外，該區域的結構會受到膽固醇的加入影響，我們推測會向螺旋的反方向扭轉，使 V36d8 與 40R1 間的距離在 10% 膽固醇加入有最短距離。另外，由 CW-ESR 光譜分析所得的距離分布顯示 29/36R1 相較於 40/47R1 有較近的分子內自旋標記距離。綜合上述光譜學所得資訊，得以一窺脂雙層中的膽固醇含量對類澱粉前驅蛋白穿膜區域結構的調節。
並列摘要
More than 90% of Alzheimer’s disease (AD) occurs sporadically without genetic linkage. Aging, hypertension, high cholesterol content, and diabetes are the well-known non-genomic risk factors of AD. The aggregation of amyloid-β (Aβ) peptides is the initial event in the pathogenesis of AD. The Aβ peptides are cleaved from amyloid precursor protein (APP) by β-secretase and γ-secretase. γ-secretase cleaves at the transmembrane region of APP without a specific cutting site, producing various lengths of Aβ peptides with different aggregation propensity and toxicity. The peptide Aβ42, which has 42 amino acid residues, is the most toxic peptide and the primary form in the Aβ aggregates in the AD brain. Since cholesterol content is one risk factor of sporadic AD, we aim to explore whether cholesterol can affect the structure of the APP transmembrane region, thereby modulating the γ-secretase cutting behavior. We synthesized a series of peptides containing the transmembrane region of APP, made spin-labeling, and identified the structural change of peptides embedded in the DOPC liposomes containing different amounts of cholesterol by double electron-electron resonance (DEER), electron spin echo envelope modulation (ESEEM), and continuous-wave (CW) electron spin resonance spectroscopy. Our results show that cholesterol decreases the structural flexibility of the APP transmembrane region and extends the transmembrane helix in the lipid bilayer. The segment of 36-40 (sequence VGGVV), which contains a Gly-Gly sequence, forms an α-helical structure rather than a 310 helix. The relative orientation of V36 and 40R1 varies in the presence of cholesterol, and the distance of these two residues is the closest in the presence of 10% cholesterol. Additionally, the distance distributions extracted from the CW-ESR spectra showed that the distance between 29/36R1 was shorter than that between 40/47R1. Together, this study provides spectroscopic evidence showing how the cholesterol content modulates the secondary structure of the APP membrane-spanning region in the lipid bilayer.
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